The broad goals of this work are to elucidate the developmental and molecular mechanisms that lead to the production of a functional central nervous system (CNS) in Drosophila melanogaster. For many animals the central nervous system is the most intricate and complex organ in the body. As the nervous system forms, not only must the various types of neurons and glia take on their identities, but once generated, these cells must send out a large number of processes and establish very precise connections on a diverse array of target tissues. For all of these steps to occur normally, and for the nervous system to develop correctly, the appropriate genes must be regulated in a precise temporal and spatial fashion within CNS cells. Knowledge of critical developmental events of neurons and glia is necessary to fully understand the prerequisites of their ultimate behavior and function. For example, a current challenge in medicine is to regenerate new neurons in damaged tissue. Presently, even in cases in which new neurons can be introduced or regenerated, it is difficult to predict their precise behavior. The identification and understanding of neural and glial specification molecules, as well as axonal pathfinding molecules and their functions, will lead to the ability to control the behavior of neurons. Not only will such research lead to new ideas and techniques for treating neuronal birth defects and lesions, but it will also contribute to our knowledge of basic processes of gene expression, cell signaling, cell connectivity and signal transduction. Ultimately, the development of the nervous system will have a profound impact on the behavior of the adult organism. This research aims to elucidate transcriptional mechanisms that lead to the specification and differentiation of the highly specialized midline cells within the Drosophila CNS. Midline cells separate the two symmetrical halves of the CNS and provide many of the signals that guide axon growth of the lateral CNS neurons. Genes expressed in the CNS midline cells ultimately control the development of these cells into both glia and neurons, and control the expression of molecules that signal to lateral CNS neurons. These experiments focus on the role of transcription factors in the regulation of gene transcription during midline cell development. To understand the roles of certain transcription factors in midline cell development, their ability to regulate genes of the midline glial lineage will be examined with mutations and ectopic expression studies. In addition, regulatory regions of midline genes will be analyzed in an effort to identify factor binding sites, as well as binding sites for other regulators of glial development. These experiments will elucidate molecular mechanisms that lead to the differentiation of midline glia. The mutation analysis and studies of regulatory regions will be conducted by graduate and undergraduate students. This research will offer students scientific training in genetics and molecular, cellular, and developmental biology that will enhance their education and prepare them for careers in the life sciences.

Overview: North Carolina State University (NCSU) faculty and students have a compelling need for a state-of-the art, high resolution confocal laser-scanning microscope (CLSM) for the simultaneous imaging of multiple gene products and their distribution in both fixed and live tissues. The only high resolution confocal on campus is a Leica SP1 Laser Scanning Microscope which was purchased in 1996. The Leica SP1 CLSM has been well maintained and while it has served the needs of faculty and students for many years, it employs outdated technology and does not meet our present and growing needs. None of the CLSMs on the NCSU campus are capable of the high resolution, high sensitivity, dynamic and fixed-specimen imaging required to monitor low levels of multiple fluorophores or to analyze their distribution within samples. This means faculty must drive to the University of North Carolina, Duke or to the Research Triangle Institute to gain access to a state-of-the-art CLSM to conduct their research. Our faculty are actively training graduate and undergraduate students and participate in high school and middle school outreach programs; however, on-campus training and outreach are constrained by the lack of a state-of-the-art CLSM. Furthermore, because of the lack of infrastructure, we are no longer competitive in hiring new faculty. A number of strong job candidates have specifically noted the lack of suitable imaging facilities as a major determinant in their taking jobs at other universities and turning down offers by NCSU. Instrumentation: A Leica SP5 CLSM. The Leica SP5 CLSM can monitor dynamic changes in the spectral properties of specimens as well as the subcellular distribution of low levels of multiple fluorophores not detectable with the Leica SP1 CLSM. Furthermore, a Leica SP5 will enable us to monitor protein-protein interactions using Fluorescence Energy Transfer (FRET) and dynamic changes in protein redistribution in response to stimuli using Fluorescence Recovery After Photobleaching (FRAP). Neither FRET nor FRAP are possible with the Leica SP1. Broader Impacts: A new CLSM is essential to reveal the biological relevance of ongoing cellular, biochemical and genetic studies at NCSU. Impacted projects include but are not limited to research focused on: genes that regulate cellular development, particularly stem cells, in both animals and plants; new molecules involved in neuron-glial interactions; mechanisms regulating protein distribution and signaling in plants in response to abiotic and biotic stresses, and mechanisms regulating wound repair in animal cells. The new CLSM will be housed in the Cellular Molecular Imaging Facility (CMIF), a University-wide facility that serves as a focal point for imaging training and research. The CO-PIs will be the co-directors of the facility. Dr. Eva Johannes, the Assistant Director of the CMIF, will maintain and manage the equipment and supervise and train new users as well as teach the course on imaging. The CLSM will have immediate and broad impacts on the training of undergraduates, graduates and post graduates at NCSU as well as undergraduates at Meredith College (near-by woman?s college) who take the imaging course. Furthermore, the new CLSM will be used by a diverse group of PIs who provide role models as they mentor undergraduates in independent research and participate in summer REU programs involving students from non-research I and minority universities. The new CLSM will also be highlighted during the tours and hands on activities for prospective students and local school children conducted by the CMIF. For example, CMIF is a focal point of the ?Expanding your Horizon? program aimed at attracting 7th grade girls from around the State of North Carolina to a career in science, mathematics and engineering.

One of the fundamental issues in developmental neuroscience is to identify molecular and cellular factors that control generation of various types of neurons and glia within the nervous system. Defective neurons and glia have been implicated in major aging-related neurological and psychiatric disorders, such as epilepsy, Huntington's, Alzheimer's, and Parkinson's, schizophrenia, and depression. The ability to restore neuron and glial cells within the nervous system will aid therapies for central nervous system diseases and injuries. Ultimately, identification of genes involved in neuron and glial development and neuron-glial interactions may provide cues for restoring function within the nervous system.

This supplement is requested to: 1) facilitate the completion of one of the major research aims of the project and 2) provide research educational opportunities for an undergraduate student. One of our major goals is to understand how midline genes are activated during development. We would like to know how genes expressed in midline glia are differentially regulated compared to genes expressed in midline neurons. For this purpose, we are currently identifying regulatory regions for several genes expressed in midline glia. Our progress could be accelerated with the help of a hard-working, undergraduate researcher. This project should be beneficial for the undergraduate student, Anna Ervin, as well. She has a very strong academic record and is very interested in obtaining research experience during the summer months. While in the lab, Anna will learn many new techniques, how to design and carry out experiments and how to communicate her results to others in the lab. She will also interact with several other faculty members in the Genetics Department during our Developmental Genetics research meetings. This experience will help prepare her for a successful career in science.

Our research is geared toward understanding and characterizing the central nervous system at a genetic level by identifying new genes that are essential for its development. If we can understand how the cells of the nervous system are generated during the course of normal development, we can then design methods for the maintenance and repair of neurons in patients suffering from neuron loss due to aging. A long-term goal for our study is to develop treatments for people who suffer significantly from age related diseases and CNS injuries.